Project Summary Research Advances in DNA sequencing have revealed that cancer genomes harbor chromosome rearrangements of unexpected frequency and staggering complexity. Despite the recognition that these variations can enable cancer development by inducing pro-growth genetic change and facilitating clonal evolution, the instigating factors and mechanisms behind rearrangement are often unknown. This proposal aims to identify the underlying causes of cancer-associated chromosome rearrangements by focusing on errors during mitosis, especially in the context of dicentric chromosome formation. Telomere fusions, which occur during human tumorigenesis when critically short telomeres become dysfunctional, generate dicentric chromosomes. This stage of telomere fusion, genomic instability, and frequent cell death is known as telomere crisis (TC). Dr. John Maciejowski's previous work has shown that the dicentric chromosomes formed during TC are resolved after attack by the cytoplasmic nuclease, TREX1, yielding chromothripsis (chromosome shattering) and kataegis (clustered hypermutation). Here, Dr. Maciejowski will use his established, genetically tractable model of TC to determine if enzymatic attack is directly responsible for observed genomic variants (Aim 1). This approach will utilize whole genome sequencing to assess rearrangement and mutation phenotypes associated with loss of a specific gene. In addition, he will develop novel assays to detect prior TC in cancer genomes (Aim 2) with the overall goal of defining the role of TC in cancer etiology (Aim 3). Finally, he will use his previously developed karyotype-based rearrangement screening and whole genome sequencing pipeline to identify additional causes of genome rearrangement by defining the genomic changes associated with dysfunction of the spindle assembly checkpoint, a cell cycle checkpoint that ensures high fidelity chromosome segregation during mitosis and is often dysregulated in cancer (Aim 4). Collectively, this proposal combines the versatility of mammalian tissue culture genetics and the power of whole genome sequencing with the aim to provide deep insights into a key aspect of tumorigenesis: the genome rearrangements that spur cancer progression and tumor evolution. Candidate Dr. Maciejowski's long-term goal is to understand the impact of errors in chromosome segregation on chromosome rearrangement, aneuploidy, and cancer development. He plans to use whole genome sequencing to assess genomic instability after the introduction of chemical or genetic perturbations in chromosome segregation or DNA repair pathways. His background in Mathematics and extensive training in chromosomal biology, the mechanisms of mitotic chromosome segregation, and human tissue culture systems provide him a solid foundation to achieve this goal. During the K99/Mentored Phase, Dr. Maciejowski will be trained in bioinformatic methods with applications in whole genome sequencing analysis. This will be critical for his future research and help launch his career as an independent investigator. Environment During the K99/Mentored Phase, Dr. Maciejowski will be supported by an outstanding group of scientists: Dr. Titia de Lange is an expert in the consequences of telomere dysfunction and will serve as primary mentor. Dr. Marcin Imielinski is a computational biologist with expertise in developing algorithms to probe cancer genome structure. Collaborators will include Dr. Peter J. Campbell, a leader in cancer genomics, and Dr. Achim Jungbluth, an expert in pathology. Together, this group comprises a strong multidisciplinary research team that will successfully execute the proposed experiments and advance a training plan that will help Dr. Maciejowski transition to an independent academic position. Dr. Maciejowski will be trained in advanced sequencing approaches including: paired end sequencing, inferring copy number from read depth, and identifying rearrangements and mutations. Career development will also be emphasized. Research will primarily be performed at The Rockefeller University, which offers unsurpassed resources, established mechanisms for career development, and a collegial academic environment. Given the strong support of his mentoring team and the excellent environment at RU, Dr. Maciejowski expects to identify and transition to an independent tenure track position at an institution supportive of his R00/Independent Phase research goals. !